Electrode for measuring concentration of choline and its esters

ABSTRACT

1. AN IMPROVED ELECTRODE FOR MEASURING THE CONCENTRATION OF THE CATIONS OF CHLORINE AND ITS ESTERS IN AN AQUEOUS SOLUTION COMPRISING: (A) AN ELECTRICALLY INSULATING BODY HAVING AN OPENING AT A PORTION THEREOF; (B) A FUSED MEMBRANE DISPOSED IN COVERING RELATIONSHIP ACROSS THE OPENING, THE MEMBRANE COMPRISING; (I) A POLYVINYLCHLORIDE MATRIX CONTAINING, (II) AN ION EXCHANGE MATERIAL HAVING THE FORMULA ((CH3)3NCH2CH2OR)+(B(C6H4X)4- WHEREIN X IS CHLORINE OR PHENOXY, R IS HYDROGEN OR -CO-R&#39;&#39; AND R&#39;&#39; IS LOWER ALKYL OR PHENYL, AND (III) A SUITABLE SOLVENT-PLASTICIZER, (C) AN INTERNAL REFERNCE ELECTODE CONTAINED WITHIN THE BODY OF (A) AND IN ELECTRICAL CONTACT WITH THE MEMBRANE OF (B).

Oct. 8, 1974 BAUM ETAL 3,840,452

ELECTRODE FOR MEASURING CONCENTRATION OF CHOLINE AND ITS ESTERS 2Sheets-Sheet l I Filed June 23, 1972 Oct. 8, 1914 BAUM EI'AL G.ELECTRODE FOR MEASURING CONCENTRATIO OF CHOLINE AND ITS ESTERS FiledJune 23, 1972 2 Sheets-Sheet a LLI Z .J O 5 LIJ 2 E 3 LL] I 2 V 2 3 3 Im O I'- Z U 2 L .1 l-

0 Lu wr 5 (J I i 2 0: 2 2 t 3" 3 m D O 4 0 CD I O 00 U) L l l I l I l fO O O O O O O O 10 I!) O l0 O '0 O N (\J m I I l I I LOG United StatesPatent 3,840,452 ELECTRODE FOR MEASURING CONCENTRATION OF CHOLINE ANDITS ESTERS George Baum, Coming, and Merrill Lynn, Big Flats, N.Y.,assignors to Corning Glass Works, Corning, N.Y. Filed June 23, 1972,Ser. No. 265,778 Int. Cl. GOln 27/46 US. Cl. 204-195 M Claims ABSTRACTOF THE DISCLOSURE Electrode for measuring the concentration of cholineand its esters in an aqueous solution wherein the sensing portion is apolyvinyl chloride membrane containing a substituted tetraphenylboratedissolved in an organic solvent which also plasticizes the PVC. Methodsof preparing and using the electrode are disclosed.

BACKGROUND OF THE INVENTION Electrodes for determining ionconcentrations in aqueous solutions are well known. Electrodes have beendesigned to measure both cationic and anionic concentrations. To measuresuch concentrations, two electrodes are commonly used; a sensorelectrode and a reference electrode. In use, the two electrodes areconnected to an electrometer and then immersed in an aqueous testsolution. Depending on the construction of the sensor electrode and theextent of ionic activity in the test solution, an electrochemicalpotential is developed. This results in a potential reading on theelectrometer. Since ionic activity is a meassure of ionic concentration,the potential reading on the electrometer can be translated into ameasure of ionic concentration in a given test solution.

The choice of sensor electrode depends on the type of ion concentrationto be measured. For cationic concentration measurements, the sensorelectrode must be sensitive to cationic activity; where anionicconcentrations are to be determined, the electrode must be sensitive toanionic activity. For a sensor electrode to be usefully sensitive to theactivity of a particular ion, the electrode should be of such a naturethat it senses the activity of that particular ion in preference to theactivities of other ions which may be present in a test solution.

The preference of a sensor electrode for certain ions is commonlyreferred to as the selectivity of the sensor electrode for certain ionsover other ions. This selectivity is governed by the tendency of thesensitive portion of the sensor electrode to sense given test ions overother ions at the same concentration. Thus, if the sensitive portion ofthe sensor electrode is of such composition as to sense more readily thetest ion activity, the EMF noted on the electrometer will be mainlyattributable to the test ion activity. This, in turn, provides anindication of test ion concentration.

Ideally, ionic concentration is related to ionic activity and EMFthrough the following relationship:

EMF=Ef-%ln A=E+ log A 0.

where EMF is the electrode potential, E is a constan, n is the ioncharge (i) and A is the activity of the specific ion in solution. Fromthe above equation, it can be seen that, ideally, a change in activity(A) equivalent to one order of magnitude causes a 59 mv. potentialchange when the ion is univalent and about a mv. or 20 mv. change,respectively, when the ion is bivalent or trivalent. Thus, since achange in EMF represents a change in test ion activity, and since testion activity can be releated to test ion concentration, a noted changein EMF can be used to indicate test ion concentrations.

3,840,452 Patented Oct. 8, 1974 When a second ionic species to which theelectrode will also respond is present, the observed potential can bedescribed by the relationship where B is the activityof the secondaryion and K is the selectivity ratio. It is desirable to constructelectrodes so that K is small in order to reduce the interference of Bwhen measuring the activity of A.

As noted above, the ion sensitive portion of a sensor electrodedetermines which ion concentrations can be measured. Typically, the ionsensitive portion is in electrical contact with an electrometer. Thesecond electrode used, a standard reference electrode such as a standardcalomel electrode (SCE), is also connected to the electrometer. When thesensor electrode and reference electrode are immersed in a testsolution, changes in EMF will generally be attributable to changes inthe activity of a particular ion which is sensed by the sensitiveportion.

Sensor electrodes are available to measure a wide variety of inorganicions such as H Na+, K+, Ag+, Cu+, Ca+ F", Cl-, Br, 1-, and NO Recentlythere has been disclosed a sensor electrode which can measure theconcentration of the organic cations of choline and its esters.

Choline, trimethyl (Z-hydroxylethyl) ammonium hydroxide, [(CH NCH CH OH]OH is an important nutritional substance. It is very soluble in waterand absolute alcohol. It is stable in dilute solutions, but inconcentrated solutions tends to decompose at a temperature of about C.Frequently, choline is used in the form of its salts, one of the mostcommon being the chloride, [(CH NCH CH OH]+Cl-. The esters of cholineare of great physiological interest, especially acetylcho line chloride[(CH NCH CH OCOCH ]+Cl-, which is thought to be essential in thetransmission of nerve impulses. Because choline and its esters oftenoccur in relatively low concentrations, and because of the interest inknowing their concentrations, attention has been directed towardproviding a satisfactory electrode for measuring the concentration ofcholine and its esters. The present invention represents a significantimprovement over an earlier electrode sensitive to choline and itsesters. The earlier electrode is discussed uinder the heading, PriorArt.

PRIOR ART Some of the earlier and present ion sensing electrodes utilizean electrode having a sensor portion consisting of a glass material.Such glass electrodes are well known and have been used to measure theconcentration of such ions as H+, Na+, and K+. Examples of suchelectrodes can be found in US. Pat. No. 2,829,090 issued to G. Eisenmanet al.

A more recent type of electrode utilizes a sensor portion consisting ofan organic liquid membrane at which ionic exchange occurs through aninterface between the organic ion exchange material and a test solution.By choosing an appropriate organic sensing phase for the organic ionexchange material, electrodes can be constructed to measure a widevariety of ions such as Ca++, K+, and the like. Examples of suchelectrodes may be found in US. Pat. No. 3,429,785 issued to J. W. Rossand US. Pat. No 3,598,713 issued to G. Baum and W. Wise. Since ionsensing in the above electrodes takes place at an aqueousorganic phaseinterface, various methods have been de vised to minimize leakagebetween the aqueous and the organic sensing phases. In U.S. Pat. No.3,438,886, issued to Ross, and US. Pat. No. 3,449,032, issued to Settzoet al., there are disclosed various porous hydrophobicorganophilicmembrane materials which can be used to separate the organic and aqueousphases while still permitting an ion sensing interface. By minimizingorganic phase to aqueous phase leakage, the above disclosed membrancestend to prolong sensor electrode life.

The above disclosures relate to electrodes for measuring inorganic ions.A more recent disclosure, US. Pat. No. 3,632,483, assigned to the sameassignee as the present disclosure, describes an electrode fordetermining the concentration of the organic ions of choline and itsesters. The sensing portion of that electrode utilizes a liquid organicsensing phase consisting of a substituted tetraphenylborate dissolved ina suitable organic solvent. The electrode has demonstrated highselectivity for choline and its esters in the presence of such ions asN'', K and Ca++ and it can measure the concentration of choline and itsesters at relatively low levels. Although that electrode has been usedin applications such as the determination of cholinesterase activity inserum, a number of operating difficulties have limited its overallutility. For example, to prevent poisoning of the electrode by exogenousproteins in serum, it is usually necessary to provide a cellophanebarrier between the porous membrane support for the organic phase andthe test solution. This cellophane barrier must be replaced at regularintervals. Often the cellophane traps an air or water pocket, leading toerratic electrode behavior. Further, it has been found that the poroussupport membrane, commonly made of glass frit, is easily plugged orpartially plugged, thus leading in time to an electrically noisy andunstable electrode.

It should be pointed out, however, that such problems also have beenassociated with other liquid organic ion exchange electrodes that havebeen used to measure ion concentrations in serum. For example, eventhough the cellophane and the porous discs used for known liquid ionexchange electrodes can be replaced periodically, the new membranes mustbe first carefully saturated with the exchanger solution and thenequilibrated with an electrolyte solution.

Because of these common problems associated with electrodes having aliquid organic phase, further attempts have been made to improve theoperating characteristics of such electrodes by modifying the membraneinterface at which ion sensing occurs.

One of the more recent publications describing such attempts is anarticle by G. J. Moody et al., A Calcium- Sensitive Electrode Based on aLiquid Ion Exchange in a Poly(vinylchloride) Matrix, Analysis 95, 910(1970). In the above article, methods are disclosed for preparing anelectrode having an ion sensitive membrane of polyvinylchloride whichcontains an ion sensing organic phase for sensing Ca++ activity. Thesensing phase is a liquid ion-exchanger incorporated in a PVC matrix.Various operational advantages are disclosed for the PVC membrane. Oneof the more significant advantages disclosed was an operational lifetimeof greater than 18 months. Another cited advantage for the disclosedpolymer membrane is that the need for an inert support material isavoided. Consequently, any ill-defined interface problems associatedwith such supports are minimized.

A recent publication by T. Higuchi et al. in Anal. Chem. 42, 1674 (1970)discloses the use of plastic membrane electrodes for measuring theconcentration of such organic ions as the tetrabutylammonium ion and thetetraphenylboron ion. In that disclosure, the sensing plastic membranesdisclosed are used for measuring the concentrations of relativelyhydrophobic organic cations and anions. The membranes prepared byHiguchi et al. consist only of a plasticized polyvinylchloride film, andno ion-exchange salt is present.

We have now found that a polymer membrane can be made which will measurethe concentration of hydrophilic organic ions; namely, the ions ofcholine and its esters.

SUMMARY OF THE INVENTION We have prepared an improved electrodesensitive to choline and its esters wherein the improvement consists ofa polymer membrane of plasticized polyvinyl chloride containing adissolved salt sensitive to choline and its esters. The electrodecomprises an insulating body having an opening at a portion thereof anda membrane disposed in covering relationship across the opening.,Themembrane comprises a polyvinyl chloride matrix containing a substitutedterephenylborate salt which is dissolved in a solvent which is asuitable plasticizer for the polyvinyl chloride plastisol from which themembrane matrix is formed. The membrane has two essentially distinctfaces, an outer face and an inner face. The outer face can be contactedwith the aqueous test solution with the contacting portion forming aninterface where the ion exchange occurs. The inner face of the membraneis in electrical contact with an internal reference electrode containedwithin the insulating body and electrically insulated from the aqueoustest solution. The internal reference electrode may be of anyconventional type, such as that comprising a silver chloride bead on asilver wire with which an electrolyte salt bridge may be used. Analternative construction involves coating a conducting metal wire suchas copper or silver with the membrane. The metal wire then serves as theinternal electrode.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1 represents a partial crosssectional view of a representative electrode formed according to theprinciples of the present invention.

FIGS. 2 and 3 represent cross sectional views of representativemodifications of the lower portion of FIG. 1.

FIG. 4 is a graph comparing the potential response curves of anelectrode of the present invention for selected ions of varyingconcentrations.

SPECIFIC EMBODIMENTS The main body or housing of our electrode may besimilar to most of the electrode bodies found useful for containing theion sensitive organic phase of liquid organic ion exchange electrodes.Such bodies are well known and they have a variety of configurations.Generally, they consist of an elongated tube of an insulating materialsuch as glass or plastic having openings at each end. One opening servesto admit and hold in place a lead from an internal reference electrodecontained within the tube. The other opening serves as a passageway forion exchange between an aqueous test solution and an ion sensing phasealso contained within the tube. In the case of known electrode bodieshousing an ion sensing liquid organic phase, a porous membrane,preferably hydrophobicorganophilic, is used to cover the sensing openingof the electrode body, thereby mechanically supporting the organicliquid phase and minimizing or preventing leakage between the aqueoustest phase and the organic sensor phase.

In our electrode, the organic sensing phase for choline and its estersis contained within the body of a polyvinyl chloride membrane which alsoserves to keep the aqueous test phase separate from the salt bridgesolution of the internal reference electrode. To prepare a satisfactorymembrane for our electrode, we have found that only certain groups ofcompounds can be used as suitable plasticizers for a PVC plastisol resinsince such compounds must also serve as effective solvents for thesubstituted tetraphenylborate salt needed to sense the activity ofcholine and its esters. Thus, such compounds, which may be designated assuitable solvent-plasticizers must serve two functions. Firstly, theymust be eifective solvents for the substituted tetraphenylborate sensingsalt such that he ion sensing salt demonstrates choline ion selectivityin the presence of other ions. Secondly, they must also be effective inplasticizing a PVC resin to form a structurally sound, easy-to-make PVCmembrane containing the sensing salt in a solvent-plasticizer solution.

It has been found that there are three groups of compounds whichaccomplish the above two goals and hence make good solvent-plasticizers.The first group includes aromatic diesters of the following formula:

COOR

COOR

wherein R and R represent alkyl groups having 4 to 18 carbon atoms. Whenalkyl groups below 0.; are utilized, the compounds are not goodplasticizers for the PVC resin and tend to result in undesirably brittlemembranes. When the alkyl groups are above C the compounds have a highmelting point, and tend to be waxy or solid, thus having viscositieswhich make it difiicult to form the membranes. Because the viscosity ofthe solventplasticizers is important for both forming the membrane(where it must be low enough to facilitate PVC membrane formation) andsolvating the sensor salt (where it should be high enough to minimizesensor phase-aqueous phase leakage) our preferred alkyl groups for thesesolvent plasticizer compounds have between 4 and 12 carbon atoms, thetotal of both alkyl groups preferably not exceeding 24 carbon atoms. Ourespecially preferred aromatic diester is dibutylphthalate. Aromaticmonoesters are to be avoided as it is thought they are too volatile toserve as effective solvent-plasticizers.

A second group of compounds which serve as good solvent-plasticizersinclude trialkylphosphates which, as in the case of the aromaticdiesters, have alkyl groups ranging from C to about C for the samereasons. Preferably, the trialkylphosphate alkyl groups are between Cand C to provide a high enough viscosity to minimize leakage yet lowenough viscosity to permit easy PVC membrane formation. Examples of suchtrialkylphosphates which serve as good solvent-plasticizers aretrioctylphosphate and tributylphosphate.

The third group of compounds which serve as good solvent-plasticizersare nitroarornatic compounds of the following formula:

wherein R is a member selected from the group consisting of hydrogen, analkyl group of 1-14 carbon atoms, lower alkoxy, and alkylcarboxy, and R'is a member selected from the group consisting of hydrogen and an alkylgroup having less than 4 carbon atoms. As in the case of the othersolvent-plasticizers, the nitroaromatic compound limitations are basedprimarily on viscosity considerations which determine the lower andupper limits for the molecular weights and chain lengths of substitutiongroups. Again, too low a viscosity will tend to promote undesirableleakage between the aqueous phase (test solution) and the sensor phase.Too high a viscosity hinders formation of a good membrane matrix fromthe PVC plastisol. Typical nitroaromatic compounds which act as goodsolvent-plasticizers are p-hexylnitrobenzene and pnitrophenyl octylether.

Suitable resins which may be used for membrane formation include manyvinyl resins which are commercially available. Examples of such resinsare the high molecular weight predominantly vinyl chloride polymers suchas Bakelite Vinyl Dispersion Resin QYOH2, Geon l2l, and Exon 654. Apreferred resin consists of polymer units having an average molecularweight of about 50,000-100,000.

The ion exchange materials for our electrode mem brane are identical tothose disclosed in US. Pat. No.

6 3,632,483. Such materials are substituted tertaphenyl boratesrepresented by the following formula:

wherein X is chloride or phenoxy, R is hydrogen or o H CR and R is alower alkyl group or phenyl. Such choline or choline ester sensing saltsare more fully described in the above patent, incorporated therein byreference.

Generalized steps for preparing an electrode employing the presentprinciples can be set forth as follows:

1. The substituted tetraphenylborate salt is dissolved in one of thesolvent-plasticizers such that the resulting solution comprises about.5-5% by weight salt with a preferred range being about 13% by weightsalt to assure a sufficient amount of salt for ion sensing.

2. Next the PVC plastisol resin is dispersed in the solution of 1. Theamount of resin in the dispersion may range from about 30-60% by weightbut a preferred amount of resin is about 50% by weight.

3. After dispersion of the plastisol rsein in the solution of 1, themembrane material is cast by conventional techniques so that theresulting sheet of PVC containing the solvent-plasticizer and salt willhave a thickness of between about 6-50 mils, preferably between 10-25mils. It was found that the electrochemical behavior of our membraneelectrodes is related to membrane thickness. Thin film (less than 6mils) did not act as permselective membranes (e.g., the observedpotential across the membrane was not altered by dilution of thesample). On the other hand, films exceeding 50 mils exhibited extremeelectrostatic sensitivity and their electrochemical characteristics weredifficult to examine. These effects are believed to be related to theresistance of the membrane. The membrane formation and control of itsthickness may be accomplished via such conventional techniques as with adoctor blade or mold, or by rolling, pressure molding, or extruding themembrane material.

4. Once the membrane material has been cast, it is fused for a shorttime at a temperature between about -240 C. with a preferred temperaturebetween 200 C. for about two minutes.

5. Once fused, the membrane material can be cut to shape in the form ofa disc which will serve as the ion sensitive membrane covering anopening of the electrode housing. The cut disc may be held in place overthe sensing opening of the electrode housing by any conventional meanssuch as with an O-ring, or by gluing to the outer rim of the openingwith a cement (e.g., a water insoluble adhesive such as an epoxy,urethane or cyanoacrylate adhesive) which will not interfere with theion sensing function of the membrane. By forming the membrane materialin sheets larger than that actually needed for the disc, a number ofadvantages are seen. For example, since the sheets can be stored, addedmembranes can be cut as needed for replacement. Further, by attachingthe disc to the electrode housing in a removable manner (e.g., with anO-ring or the like) the membrane can be easily disposed of before movingfrom test solutions of greatly different chemical composition. Since theformation of the membrane sheet is a relatively easy and inexpensiveprocess, it may be more economical to use a new disc for eachmeasurement even though one of the primary advantages. of the presentmembranes is their relatively long sensor life compared to known liquidorganic sensing phases.

The actual construction of our electrodes can be better understood byreferring to FIGS. 1-3. In FIG. 1, an electrode 3 consists of a palstichousing 5 in which is contained an internal reference electrode 7 heldin place by a housing cap 9 which is of an insulating material such asrubber. The housing cap 9 can hold the internal reference electrode 7 inplace by friction or other means.

A lead 11, in electrical contact with the internal reference electrode 7and insulated by an insulating material 13 extends from the electrodecap 9 and is connectable to an electrometer. Contained within theelectrode housing is a conventional salt bridge solution 15 inelectrical contact with the internal reference electrode 7 and themembrane 17. The membrane 17 is held in place by an internally threadedplastic cap 19 which engages mating threads on the lower portion of thehousing 5. The membrane 17 is further secured with the aid of an O-ring21 which helps retain the membrane during assembly. A

similar O-ring 23 may be used to assure a tight fit of the cap 19against the housing 5.

The outer face of the membrane 17 provides an interface for ion sensingthrough an opening 25 in the membrane retaining cap 10. The inner faceof the membrane 17 is in electrical contact with the salt bridgesolution 15.

FIGS. 2 and 3 illustrate two other ways the membrane 17 may be attachedto the sensing ends of glass electrode housings 31 which do not requirethreaded portions to engage a threaded cap such as that shown in FIG. 1.In FIG. 2, the mmebrane 17 is shown held in place by a nO-ring 27. InFIG. 3 the membrane is shown held in place by a suitable cement 29.Other methods for attaching the membrane 17 to the sensing end of anelectrode housing are, of course, possible. The main requirement is thatthe membrane act as a barrier supporting the salt bridge solution, whenused, thereby keeping its from leaking into the test solution or viceversa.

Our initial membranes were prepared by using as solvent-plasticizers theacetylcholine exchanger solvent systems described in U.S. Pat. No.3,632,483. Although functional electrodes resulted, it was found thatthese earlier polymer membranes (films) had poor mechanical propertiessuch as low tear strength and a limited useable lifetime. After aboutl-2 weeks of use, the performance of the electrodes deteriorated andthey could no longer be used. Other solvent-plasticizers were thenconsidered to find which solvent-plasticizers would accomplish best thetwo-fold purpose as solvent for the borate salt and as plasticizer.Dioctylphthalate, di-isononylphthalate, and mixed systems such as thosecontaining 50% (wt.) 1,2- dimethyl-3-nitrobenzene gave operablemembranes but without the desired long life. However, whendibutylphthalate was used as a solvent-plasticizer for the ion exchangesalt and the PVC, the resulting membranes were found to have excellentelectrical stability, a long life, and generally excellent overallelectrode characteristics. An added advantage found in using thedibutylphthalate solvent-plasticizer was that operable membranes couldbe cut from polymer sheets which had been stored for several weeks. Incontrast, it was found that membranes prepared With dioctylphthalate hadshorter life times when those polymer sheets were stored for a period oftime.

Although the membrane preparation is subject to various modifications,our membranes are preferably prepared as plastisols by incorporating theexchanger salt solution in a vinyl dispersion resin. Vinyl dispersionsare suspensions of resin in liquids which do not dissolve the resin atordinary temperatures. In a plastisol, the vinyl resin particles aredispersed into a plasticizer, the mixture is cast, drawn, or sprayedinto a thin film, and then the mixture is heated to fuse the particlesand form a continuous film. In our membranes the salt dissolved in thesolvent-plasticizer becomes an integral part of this film.

Other less desirable methods for utilizing resins require a solution ofthe resins at low concentrations or the manipulation of the resin in amolten state. The first method places severe limitations on the type andmolecular weight of the resins that can be utilized, and the secondmethod requires undesirably high temperatures, and the use of expensiveequipment. The vinyl dispersion technique avoids these limitations andpermits the use of high molecular weight vinyl chloride homopolymerswhich cannot be used in the solution process. These higher molecularweight homopolymers produce strong, flexible films when used asplastisols.

Not all of our solvent-plasticizers are commonly used as plasticizersfor PVC. For example, in one of our initial membranes, 3-nitro-o-xylenewas used as the solventplasticizer. Although a functioning electrodemembrane resulted, the performance tended to deteriorate in time and themembrane became embrittled. Higher quality membranes were later producedusing some of the more conventional plasticizers for the PVC which wouldalso serve as suitable solvents for the substituted tetraphenylboratesalt. After preparing our ion sensing membranes with varioussolvent-plasticizers, it was found that one of the bettersolvent-plasticizers was dibutylphthalate. In the examples below,electrodes having a membrane plasticized with that diester were used andspecific directions for the preparation and use of such electrodes aregiven. It is intended that the following examples should be illustrativeonly as to the preparation and use of a particularly preferredelectrode.

EXAMPLE I Preparation of Membrane A 5% by weight solution ofacetylcholine tetra (pchlorophenyl) borate in dibutylphthalate wasprepared. This solution was used as the plasticizer for the plastisoltype PVC powder, Vinyl Dispersion Resin QYOH-Z (Union CarbideCorporation). A paste of 50% by weight of the solvent-plasticizer wasmixed well with the plastisol PVC powder. The paste was spread in a moldsuch that the thickness of the resulting membrane would be between 10-25mils and heated to 200 C. for two minutes. The resulting membrane wasremoved after cooling to room temperature. A small disc, smaller thanthe diameter of the electrode barrel was cut from the sheets.

Construction of the Electrode The above disc (0.232 inch diameter) wasplaced into the bottom cap of a membrane assembly device for attachingmembranes to electrode barrels. A small O-ring was placed behind thedisc to prevent twisting of the membrane when the electrode assembly wasto be screwed together. See FIG. 1. The chamber behind the disc (e.g.,inside of the 'barrel) was filled with a saturated KCl-AgCl solution andan Ag/AgCl electrode was placed in contact with the internal electrolytesolution. Although a double junction internal reference electrode orsimilar internal reference electrode can be used, all of ourmeasurements were made with the Ag/AgCl electrode dipped directly intothe internal electrolyte. All potential measurements were made against aFisher 13-639-57 cracked bead calomel reference electrode, hereinafterreferred to as S.C.E. The electrometer used for making the indicatedmeasurements was a Corning Model 12 pH meter.

TABLE I.RESPONSE TO SELECTED IONS (mv. vs. S.G.E.) K"

Acetylcholine Selectivity ratios, calculated from simple electrolytesolutions at 0.1 M concentration.

relationship given by the first equation discussed under Background ofthe Invention.

EXAMPLE II An electrode having the dibutylphthalate-plasticized membranewas also used to determine the activity of serum cholinesterase bymeasuring the rate of change in concentration of a known amount ofacetylcholine after addition of known volumes of serum containingcholinesterase. Cholinesterase is an esterase enzyme which hydrolyzesesters of choline. The rate of change of acetylcholine concentration wasmonitored by recording the output from an expanded scale pH meter (theComing Model 12) on a mV full scale recorder using a mercury cellpowered zero suppression device. All enzymatic activity determinationsWere conducted at 25 .0 C. The activity was calculated from the slope inthe 1-55% hydrolysis region of the enzymatic hydrolysis of acetylcholineby the enzyme acetylcholinesterase. Five quantities of serum containingcholinesterase were added to solutions containing known amounts ofacetylcholine. In each case, the activity of the serum cholinesterasewas determined and expressed in activity units wherein 1 unit designatesthe hydrolysis of one micromole of acetylchlorine per minute at pH 8.0and 25 C. These results are given in Table 11.

TABLE II Units/activity found Units/m1.

Volume/serum added (ml.):

As can be seen from Table II, the determined activities of the fivesamples, expressed in units per ml., were found to be within a range of0.065 to 0.070, thus indicating a relatively high degree of reliabilityfor the electrode when used for determining acetylcholine concentrationsand, consequently, cholinesterase activity.

EXAMPLE III Comparison With Other Methods TABLE IIL-COMPARISON OFACETYLCHOLINESTERASE ASSAY METHODS Colorimetric Polymer Liquid proceduremembrane membrane 5.1) 5.9 10.8 32.3 Percent C.V 5. 4 7. 9 23 Norm-Alldata are expressed in International Units (I.U.) per mg. X=averageactivity; S.D.=standard deviation; Percent C.V.=percent coeflicient ofvariation.

As can be seen from Table III, both the liquid and polymer membraneelectrodes yield significantly higher activity values than thecolorimetric (Hestrin) procedure.

However, it has been pointed out that initial rate activity values foracetylcholinesterase are about 20% higher than is commonly obtained bythe Hestrin method which is conducted at conditions leading to 15% and40% hydrolysis. In our method, activity values are obtained during 110%hydrolysis. Further information regarding the Hestrin colorimeterprocedure may be found in an article by S. Hestrin, J. Biol. Chem., 180,249 (1949) and an article by L. T. Kremzner and I. B. Wilson, J. Biol.Chem, 238, 1714 (1963).

In the above experiments it was found that the performance of ourpolymer membrane electrode was markedly superior to the preferred liquidmembrane electrode of US. Pat. No. 3,632,483. The polymer membraneelectrode has a very short recovery time between assays (1-3 minutes)and the start-up time after an overnight storage is only about 10minutes. The results obtained with the polymer electrode exhibitsignificantly less scatter than the results obtained with the liquidmembrane electrodes.

EXAMPLE IV Further Assays Our electrode was also used for further assaysof partially purified acetylcholinesterase solutions. Aliquots of astock solution, containing approximately 0.5 mg./ml. ofacetylcholinesterose were assayed with our membrane electrode procedure.The activity of the solution expressed in I.U., was calculated by knownmeans. The data obtained are given in Table IV. As can be seen fromTable IV, the response of the electrode is linear with the volume ofenzyme solution added for over a one decade range of volumes.

TABLE IV Assay of Acetylcholinesterase Solution By using the cholinesensing membrane of our electrode, numerous advantages have been foundover the use of the liquid ion exchange electrode. One of the moresignificant advantages is that a protective cellophane film is notrequired for our polymer membrane when serum choline determinations aremade. This provides not only a simplification in construction, but alsoa major improvement in reliability since many operational difficultieswere attributed to the cellophane film, as noted above.

It should be pointed out that although our polymer membrane electroderepresents a significant improvement over those electrodes described inUS. Pat. No. 3,632,438, many of the teachings in that patent areapplicable to the present electrode. One of the more importantcomponents for our choline sensitive membranes is thesolventplas'ticizer used to plasticize the PVC and solvate the cholineion sensing salt. The detailed examples given were based on using ourpreferred solvent-plasticizer, dibutylphthalate. Othersolvent-plasticizers (e.g., p nitrophenyl octyl ether and those of thetypes listed above) were also found to result in operable choine sensingmembranes. Accordingly, since our polymer membrane electrode is subjectto numerous variations, all within the scope of the present invention,it is intended that the examples given above should be construed asmerely illustrative and not limiting.

and 'R' is lower alkyl or phenyl, and (iii) a suitablesolvent-plasticizer,

(c) an internal reference electrode contained within the body of (a) andin electrical contact with the membrane of (b).

2. The electrode of claim 1 wherein the solvent-plasticizer comprisesaromatic diesters of the formula -COOR COOR wherein R and R are alkylgroups each having 4 to 18 carbon atoms.

3. The electrode of claim 2 wherein the aromatic diester isdibutylphthalate.

4. The electrode of claim 1, wherein the solvent-plasticizer is atrialkylphosphate, the alkyl groups each having 4 to 18 carbon atoms.

5. The electrode of claim 4 wherein the trialkylphosphate is selectedfrom the group consisting of trioctylphosphate and tributylphosphatc.

6. The electrode of claim 1, wherein the solvent-plasticizer is anitroaromatic compound of the formula wherein 'R is a member selectedfrom the class consisting of hydrogen, an alkyl group having 1-14 carbonatoms,. lower alkoxy, and alkylcarboxy, and R is a member selected fromthe class consisting of hydrogen and an alkyl having less than 4 carbonatoms.

7. The electrode of claim 6 wherein the nitroaromatic compound isp-nitrophenyl octyl ether. I

8. The electrode of claim 1 wherein the membrane has a thickness betweenabout 10 to about 25 mils.

9. The electrode of claim 1 wherein the weight of ion exchange materialto solvent-plasticizer is about 0.5 to 5.0%.

10. The electrode of claim 1, wherein the insulating body of (a) is aplastic material, and the membrane of (b) has a thickness between about10 to about 25 mils and comprises a polyvinyl chloride matrix containingacetylcholine tetra (p-chlorophenyl) borate as the ion exchanger anddibutylphthalate as the solvent-plasticizer.

References Cited UNITED STATES PATENTS 3,632,483 1/1972 Baum 204195 L3,655,526 4/1972 Christian 204-195-' 3,691,047

9/1972 Ross et al 204-195M OTHER REFERENCES TA-HSUNG TUNG, PrimaryExaminer U.S. Cl. X.R. 204--1T, 195 L UNITED STATES PATENT OFFICE 7CERTIFICATE OF CURRECTION Patent No. s a s Dated October 8, 197

Inventor(s) George Baum and Merrill Lynn It is certified that errorappears in the above-identified patent and that said Letters Patent arehereby corrected as shown below:

Column 3, line 3, change "brances" to branes Column 3, line +7, change"Analysis" to. Analyst Column l, line 8, change "terephenylborate"totetraphenylborate Column 4, line 70, change "he" to the Column 6, line23 change "rsein" to resin Column 6, line 71, change "palstic" toplastic Column 7, line 16, change "10" to 19 Column 7 line 22, change"mm'e'brane" to membrane Column 7, line 23, change "a nO-ring" to anO-ring" Column 8, line 37, change "sheets." to sheet.

Column 9, line 17', change "1-5572" to 1-5% Column 9, line 2h, change"acetylchlorine" to acetylcholine Column 10, line 28, change"acetylcholinesterose" to acetylcholinesterase Column 10, line 70,change "choine" to choline Column 11, Claim 1, line 3, change "chlorine"to choline Signed and sealed this 11th day of March 1975.

(SEAL) Attest:

1 C. MARSHALL DANN RUTH C. MASON Commissioner of Patents AttestingOfficer and Trademarks

1. AN IMPROVED ELECTRODE FOR MEASURING THE CONCENTRATION OF THE CATIONSOF CHLORINE AND ITS ESTERS IN AN AQUEOUS SOLUTION COMPRISING: (A) ANELECTRICALLY INSULATING BODY HAVING AN OPENING AT A PORTION THEREOF; (B)A FUSED MEMBRANE DISPOSED IN COVERING RELATIONSHIP ACROSS THE OPENING,THE MEMBRANE COMPRISING; (I) A POLYVINYLCHLORIDE MATRIX CONTAINING, (II)AN ION EXCHANGE MATERIAL HAVING THE FORMULA((CH3)3NCH2CH2OR)+(B(C6H4X)4- WHEREIN X IS CHLORINE OR PHENOXY, R ISHYDROGEN OR -CO-R'' AND R'' IS LOWER ALKYL OR PHENYL, AND (III) ASUITABLE SOLVENT-PLASTICIZER, (C) AN INTERNAL REFERNCE ELECTODECONTAINED WITHIN THE BODY OF (A) AND IN ELECTRICAL CONTACT WITH THEMEMBRANE OF (B).